In collaboration with the laboratory of Dr, Flemming Hansen, Technical University of Denmark, we have developed a fully automated cell recognition and fluorescent focus-measuring program in the form of a macro within the image analysis program Image Pro Plus. This has been very successful in the basic tasks of detecting fluorescent foci formed by plasmids and specific chromosomal loci in cells of the bacterium Escherichia coli. As our needs for analysis of fluorescent images become more demanding, we continue to develop and adapt this system to meet them. We have developed an analytical tool "Inspect" that facilitates the analysis of images that have two color fluorescence information. In conjunction with the data collection programs, we have developed an array of spreadsheet programs to analyze and portray the data. We have completed a major new thrust in the development of the image analysis software. This allows the automated identification of multiple classes of cells in various states of division, and of foci that are dividing etc. These cells can then be inspected directly as a sub-class of images extracted for inspection. New sub-classes can then be defined and analyzed with seamless switching between the numerical data and the individual cell images. This allows us to analyze large populations in far more detail than was previously possible. This year we have expanded the utility of our automated focus measuring and analysis software. The system is now capable of recording the intensity and aspect ratio of each focus. This allows assessment of the replicational state of the DNA locus and indicates whether segregation is in its earliest phase. In addition we are re-writing the software to facilitate the analysis of triple-labeled cells and the automated detection of co-localization of double and triple labels.